Micropuncture study of nephron function in the rhesus monkey

Micropuncture study of nephron function in the

rhesus monkey

Cleaves M. Bennett, … , Barry M. Brenner, Robert W.

Berliner

J Clin Invest. 1968;47(1):203-216. https://doi.org/10.1172/JCI105710.

The function of the proximal and distal tubule was studied in the rhesus monkey during antidiuresis and during the diuresis after furosemide administration (during which extracellular fluid volume was maintained).

In the proximal tubule, fluid to plasma ratios for sodium, potassium, and osmolality

approximated unity. During antidiuresis, about 30% of the filtered water remained at the end of the accessible portion of this segment (92% of length). Fluid was hypotonic to plasma throughout the distal tubule. 25% of the filtered water was present in the early distal tubule. Small but significant water reabsorption (about 8% of filtered) occurred in remainder of this segment. Tubule fluid to plasma potassium concentration ratios tended to increase along the distal tubule, and the amount of potassium, relative to the amount filtered, increased from 13% in the early portion of this segment to 26% in the late portion.

After furosemide was administered animals excreted about one-third of the filtered sodium and water. Despite this diuresis, electrolyte and water reabsorption along the proximal tubule did not differ from values obtained in control animals. Osmolality and sodium

concentration of fluid from the distal tubule approached those of plasma. 22% of the filtered sodium (twice the control values) reached the distal tubule, whereas the fraction of filtered water remaining was only slightly increased. These findings indicate that, after […]

Research Article

Micropuncture Study of Nephron

Function

in

the Rhesus Monkey

CLEAVES M. BENNETr, BARRY M. BRENNER, and

ROBERT W. BERLINER with the technical assistance of JuLHA L. TROY

From the Laboratory of Kidney and Electrolyte Metabolism, National

Heart Institute, National Institutes of Health, Bethesda, Maryland

A BST RACT The function of the proximal and distal tubule was studied in the rhesus monkey during antidiuresis and during the diuresis after

furosemide administration (during which extracel-lular fluid volume was maintained).

In the proximal tubule, fluid to plasma ratiosfor

sodium, potassium, and osmolality approximated unity. During antidiuresis, about

30%

of the

fil-tered water remained at the end of the accessible portion of this segment (92% of length). Fluid was hypotonic to plasma throughout the distal tubule. 25%o of the filtered water was present in the early distal tubule. Small but significant water

reabsorption (about 8% offiltered) occurred in the remainder of this segment. Tubule fluid to plasma potassium concentration ratios tended to increase

along the distal tubule, and the amount of potas-sium, relativetotheamountfiltered,increased from

13%

in the early portion of this segment to

26%r,

in the lateportion.

After furosemide was administered animals

ex-creted about one-third of the filtered sodium and water. Despite this diuresis, electrolyte and

water reabsorption along the proximal tubule did not differ from values obtained in control

An abstract of this work appears in J. Clin. Invest.

1967. 46: 1035. Completetables of data from these

experi-ments areavailablefrom the AmericanDocumentation

In-stitute, 2000 P Street, N. W., Washington, D. C. 20036.

Address requests for reprints to Dr. C. M. Bennett, Laboratory of Kidney and Electrolyte Metabolism, Na-tional Heart Institute, Bethesda, Md. 20014.

Dr. Brenner is a Postdoctoral Research Fellow, Na-tional Heart Institute.

Received for publication 18 July 1967 and in revised form 15September 1967,

animals. Osmolality and sodium concentration offluid from the distal tubule approached those of

plasma.

22%o

of thefiltered sodium (twice the con-trol values) reached the distal tubule, whereas the fraction of filtered water remaining was only slightly increased. These findings indicate that, after the administration of this drug, inhibition of

sodium reabsorption occurred in the water-im-permeable segment of the nephron, rather than in theproximal tubule. After furosemide

administra-tion, all tubule fluid to plasma potassium

concen-tration ratios in the distal tubule were equal to or greater than one, suggesting inhibition of active potassium reabsorption at or prior to this site.

Fluidto plasmabicarbonate concentration ratios from the midportion of the proximal tubule were consistently less than one in normal monkeys. After acetazolamide was administered, the bicar-bonate concentration of samples of tubule fluid

recollected from these same sites was the same as,

or higher than in plasma. This fact demonstrates the inhibition of bicarbonate reabsorption in this portion ofthe tubule.

INTRODUCTION

It is likely that many of the basic transport

proc-esses operative in renal tubule cells are similar in rat, dog, and man. Indeed, micropuncture stud-ies in the dog and rat have revealed that individual nephron function in these species is quite similar (1, 2). Those differences which have been found

are primarily of a quantitative nature and are not necessarily fundamental. It has been found, how-ever, that the extent of sodium, water, and

TABLE I

Summaryof Mean Values of Glomerular Filtration Rates (GFR), Fractional Tigater, SodiumII and

Experimental (U/P) (Pa/n)

condition GFR osmolality intilin

ml/min per kg*

Hydropenia 1.45 ±0.23t 2.66 + 0.39 0.45(%O 4 0.09

(62)§ (33) (61)

Furosemide 1.50 + 0.39 0.94i0.03 32.5% +4.24

(30) (27) (28)

*_{Experimental kidney only. $Values represent mean + one standard deviation.}

carbonate reabsorption in the proximal tubule, and sodium and water reabsorption and potassium addition in the distal tubule are different in dog and rat. It is not possible to be certain whether these differences are inherent characteristics of these species or represent differences in metho-dology. Nevertheless, they serve to emphasize that extrapolation of data obtained in one species to another species must bedone with caution.

There are few studies of renal function in pri-mates other than man, primarily because it is easy

to perform clearance experiments in man. How-ever, clearances do not accurately measure func-tion at all sites along the nephron, particularly in

the more proximal portions (3). Accordingly, in the presentinvestigationthe functions of the proxi-mal and distal tubule in the rhesus monkey have been studied using the micropuncture technique both under conditions of normal antidiuresis and after the administration of furosemide and

aceta-zolamide. To the extent that nephron function in themonkeycanbeassumedtobemore representa-tive of nephron function in other primates, the

re-sults of these studies are likely to be more

perti-nenttotheinterpretationof themore indirect mea-surements of renal function in man. In addition,

since these studies indicate that micropuncture of

the monkey nephron is feasible, this animal may be a useful model for evaluating the

pathophysi-ology of renal disorders and the mechanism of

ac-tion of diuretic drugs in relation to man.

METHODS

General procedure

Studies were performed on young male and female

rhesus monkeys (Macaca niulatta) weighing 2.7-6.0 kg. All monkeys were fed a balanced diet of Purina dry monkey chow supplemented with fresh fruit, and they all were allowed free access to water. They were

de-§ Numbers in parentheses

prived of food but not water for 18-24 hr prior to study in order to reduce the volume of abdominal contents at thetime of micropuncture.

The animals were anesthetized rapidly by an intra-venous inj ection of 20 mg/kg sodium pentobarbital and were given small supplemental doses as necessary. Tracheotomy was performed in all experiments. In-dwelling polyethylene catheters were inserte(l into a fe-moral vein to administer fluid, inulin, and (Irugs, into a femoral artery for periodic samplings of blood an(l esti-mation of arterial blood pressure, and into the left com-moncarotid artery to thelevel of the aortic arch in order to inject lissamine green. The right ureter was exposed

and catheterized through a suprapubic incision. The right kidney served as the control side in all experiments. The left kidney was exposed through a flank incision, gently dissected free of its perirenal andlperitoneal at-tachments, and its ureter cannulated within 2 cm of its

origin. The animal was placed on a heated pad atop a

micropuncture table, and the abdominal wound was ex-posed by means of retraction sutures and a bulk wedge inserted under the right flank. The kidney was then sus-pended in a malleable metal holder so that it would be

snugly encased, thereby reducing pulse and respiratory

motion. A 1 cm2 portion of renal capsule was removed, the area illuminated by a quartz rod, and the exposed

surface continuously bathed with mineral oil heated to

370C.

Samples from proximal and distal tubules were oh-tained using standard collection techniques. The rate of collection was adjusted to keep a short column of oil

stationary just distal to the puncture site. Distal tubules could be identified on the surface by their relative

transparency, small size, and straight course. Occa-sionally their identification necessitated the intra-arterial injection of 0.25 cc of 8-10% lissamine green; appearance time was approximately 20-35 sec after the rapid (lye in-jection. In most instances, 15-20 nliter of fluid was ob-tained for analysis. After the fluid was collected,

punc-ture sites were localizedby means ofmicrodissection.

Sharpened micropipettes with external tip diameters of 8-12 _Ae were prepared as previously described (4). All micropipettes were externally coated with silicone

(Siliclad, Clay-Adams, Inc., New York), filled just

TABLE I (Concluded)

Potassium Excretion, and Plasma Composition during Hydropenia and Furosemide Diuresis

(lJ/P)NaX

(11PIn X1( (U'/P)

Kn Plasma

sadium

Plasma potassium mEq/liter

0.08% ± 0.07 22.8% ± 6.6 143 ± 2.5 3.2 ± 0.23

(46) (46) (60) (60)

28.5% i 4.66 81.6% i 14.4 149±4.6 2.5:1= 0.57

(20) (20) (30) (30)

indicate number of observations.

In all studies, after an equilibration period of at least 30 min following the injection of priming and sustaining inulin infusions, 15-20 min clearance periods were ob-tained. Samples of arterial blood were drawn at the

mid-point of each period, while urine from each ureteral catheter was allowed to empty under oil into volumetric containers.

Three experimental protocols were employed: Control antidiuresis. 11 monkeys were prepared for micropuncture and infused with 12.0-22.5 ml of 0.9%o

NaCl/hr. While no attempt was made either to restrict oralfluid intake prior to study or to administer exogenous

vasopressin, all animals in this group were in

antidiure-sis. In nine animals from this group, samples from both proximal anddistal tubules were obtained; in twoanimals

only distal samples were obtained.

Furosemide diuresis. Five antidiuretic animals were infused with 0.9% NaCl at a rate of 22.5 ml/hr for ap-proximately 1 hr prior to micropuncture. During this time two or three control clearance periods were ob-tained. Furosemide (3-5 nmg/kg) was given intravenously

as a prime, and the same dose was administered per hour. The animals incurred a fluid deficit of 25-50 ml. Thereafter, extracellular fluid volume was maintained at

this level by the infusion of 0.9% NaCl at a rate, esti-mated at frequent intervals, equal to the rate of urine flow.

Control antidiurcsis-.bicarbonate measurements. In four antidiuretic animals, proximal tubule bicarbonate concentration was measured using quinhydrone microelec-trodes. After control collections were made, three of these animals were given a single intravenous injection

of acetazolamide (20 mg/kg), and samples were recol-lected from the previous puncture sites for repeat

mea-surement ofbicarbonate concentration. The rise in urine flow rate was modest after the administration of

aceta-zolamide, and volume replacement was therefore

un-necessary.

Analytical

methods

The osmolality of tubule fluid, plasma, and most urine

samples was determined microcryoscopically by the

technique of Ramsay and Brown (5). When urine

vol-umes were adequate, osmolality was measured with an

Aminco-Bowman osmnometer.1 Sodium and potassium

1 American Instrument Co., Inc., Silver Spring, Md.

concentrations in tubule fluid were measured simultane-ously and in duplicate by the method of Vurek and Bowman (6) using a dual channel helium-glow ul-tramicrophotometer. Sodium andpotassium concentrations

in plasma and urine were determined with an internal standard flamephotometer.2

Inulin concentration in tubule fluid was measured by

thefluorophotometric method of Vurek and Pegram (7),

modified to increase the hydrolysis reaction time at 1000C

to 10 min. Inulin concentration in plasma and urine was determinedby the anthrone method ofFiuhr,Kaczmarczyk, and Kruttgen (8).

Quinhydrone microelectrodes, similar in design to those described by Pierce and Montgomery (9), were used to estimate tubule fluid bicarbonate concentration. In place of mercury, however, each electrode was filled with mineral oil which had been equilibrated with 5%

C02immediately prior to use. Approximately 10 nliteror

less of proximal tubule fluid or a similar volume of freshly prepared bicarbonate solution, preequilibrated with 5%o C02, was aspirated directly into the microelec-trode and the electrode tip sealed with egg albumen. A Cary vibrating reed electrometer was used to deter-mine the potential difference between the quinhydrone

microelectrode and a calomel half-cell; both were im-mersed in a saturated solution of KC1 previously

equili-brated with 5% C02 and maintained at 370C. The pH

of the samples can be calculated from the Pierce-Mont-gomeryequation:

pH=Eqh-Ecal-emf

0.0001982T

where Eqh and Ecal represent the standard potentials for the quinhydrone and KCl-calomel electrodes

re-spectively, and T=absolute temperature. Bicarbonate

concentration in tubule fluid samples was determined

from a standard curve derived from the relationship between the log (HCO3) and the measured emf. Arterial blood andurine,collectedanaerobically, wereanalyzedfor pH at 37°C in a Beckman model 76 pH meter. Plasma

C02 content was determined manometrically with a

Natelson microgasometer.

TABLE II

Measurements of Length of Nephron Segments

Species Reference Proximaltubule Loop of Henle Distaltubule

mm mm mm

Rat (I11) [9.0-13.5]* [5.0-7.3] [2.5-2.7]

(12) 10.0 i:0.31 1.4 [0.9-1.6]

Dog (13) 15.1 1: 3.3

[9.0-24.0]

(12) 14.8 [5.2-15.0]§ [3.0-3.9]

[12.8-16.5]

Rhesus

_monkeyll

This study 6.4 :1: 1.5 1.4 4 0.3§ 3.1 ± 1.0

[3.6-9.6] [0.9-1.9]§ [1.4-6.6]

Man (14) [7.2-23.1] [0-5.6]§ [1.6-4.2]

* Numbers in brackets represent the range of measurements. t Unbracketedvalues represent the mean 1 one standard deviation. §Represents measurements of the descending limb only.

11

Note that the rhesus monkeys used in thesestudies wereimmatureanimals, whereas the other speciesstudied

wereadult animals.

RESULTS

Control antidiuresis

Values of glomerular filtration rate (GFR) are

shown in Table I. A mean GFR of 1.45 ml/min per kg (±0.23 SD) was observed on the experi-mental side compared to a mean value of 1.70 ml/ min per kg (±0.29 SD) on the control side. These filtration rates closely agree with values previously reported for monkeys and other small primates in the unanesthetized state (10).

Tubule dissection. 100 superficial proximal or distal tubules with attached loops of Henle were dissected. Measurements ofthelength of these seg-ments are shown in Table II, along with compara-tive measurements from other species (11-14).

In every instance, theloopwas short and confined

to the cortex. Nephronswithlong loopsextending

into the medulla were always associated with

juxtamedullary glomeruli and were not accessible

tomicropuncture.

Often theproximal tubule remained on the

sur-face for most of its length, so that a greater per-centage of its total length was accessible to

punc-ture than in the rat and dog. Samples were ob-tained from sites in the proximal tubule ranging

from 12 to

92%o

of its total length.

After the intra-arterial injection of lissamine green, many distal tubules could be seen on the surface of thekidney. Occasionally the junctionof

two distal tubules could be seen. The cortical

col-lectingductssoformed always coursed beneath the

TABLE III

Summary of MeanOsmolality,Sodium and Potassium Concentration Ratios for the Proximal Convoluted Tubuleduring Control Antidiuresis and Furosemide Diuresis

(TF/P)osm (TF/P)N, (TF/P)K

Control Control Control

antidiuresis Furosemide antidiuresis Furosemide antidiuresis Furosemide

Mean 0.98 1.01 1.03 1.00 1.04 0.99

SD 4 0.02 4 0.03 ±0.05 i 0.03 i 0.03 i0.21

No. samples 17 20 20 14 21 14

TABLE IV

Summary of Mean Osmolality, Sodium and Inulin Concentration Ratios for the Distal Convoluted Tubuleduring ControlAntidiuresis andFurosemideDiuresis

(TF/P)osm (TF/P)N& (P/TF)ln(early D.T.)*

Control Control Control

antidiuresis Furosemide antidiuresis Furosemide antidiuresis Furosemide

Mean 0.50 0.89 0.43 0.85 0.25 0.29

SD 1 0.16 0.06 4 0.15 A 0.08 4 0.04 4 0.05

No. samples 29 11 31 11 13 6

No. animals 8 5 8 4 7 2

*_{Representssamples obtained from 24 to 49 % of the distal convoluted tubule (D.T.)}

surface tubules immediately, and thus they were not accessible to micropuncture.

Osmolality. The tubule fluid to plasma (TF/P) osmolality ratios of 17 samples from proximal tubules in six animals are shown in Ta-ble III and are plotted on a log scale as afunction

of puncture site in Fig. 1A. It can be seen that fluid collected from the proximal nephron was isosmotic with plasma (mean plasma milliosmo-lality = 307.5; range= 296-327). In contrast,

samples from the distal tubule (Table IV and Fig. 1A) were always hypotonic to plasma and remained unchanged throughout the accessible length of this nephron segment (the slope was not significantly different from zero, as shown in Table V).' The final urine to plasma osmolality

ratio during these studies averaged 2.66 +0.29

SD.

Water reabsorption. The data from control monkeys are shown in Fig. 2A. Approximately one-third of the filtered volume remained at the

end of the accessible portion of the proximal

tu-bule. The slope of the line representing the

re-lationship between the fraction of filtered water

remaining

[(P/TF)

inulin], and puncture site in this segment of the nephron is -0.00559±

0.00094 SE, and its intercept is at y=0.995.4

S_{The logs of the (TF/P) ratios (osmolality, sodium,}

and potassium) or of the clearance ratios (water,

so-dium, and potassium) were related to the location of the

puncture site in the distal tubule (expressed as a per-centage of its total length) using the method of least

squares (15). For each relationship the slope and its standarderror werecalculated,and the significance of the

relationship was determined using the Student's t test,

where t=_slope/sE.

4Thetheoretical interceptonthe y axis is at 1.0, since

the concentration of inulin in theglomerular filtrate must

equal the concentration of inulin in the plasma.

Significant net reabsorption of water was not observed in the short pars recta of the proximal tubule, in the loop of Henle, and in the inaccessible early segment of the distal tubule (Fig. 2A). Beyond this point in the distal tubule, small but

statistically significant reabsorption of water was

observed.The slopeof theregression line forthese data is given in Table V and correspondsto

reab-4.0 2.0

(TF/Psm 1_.0

0.5

% PROXIMAL 25 50 75 100

"_0VWW .... .~~~~~~~~~~~~

% DISTAL URINE 0 25 50 75 100

mi , _m

II r--l~~S

Normal Hydropenia(81 Osmololily

FIGURE 1A Tubule fluid to plasma (TF/P) and urine to plasma (U/P) osmolality ratios during hydropenia.

Inthisand subsequent figures, the number in parentheses refers to the number of animals, and the urine values

represent mean values for each animal.

2.0

(TF/FosI

0.5

F

0.25L

% PROXIMAL % DISTAL URINE

25 50 75 100 0 25 50 75 100

I T---.m-II --,r--l

m--|- | 1I 1 -, |

Furosemide (5) Osmololity

FIGURE IB TF/P and U/P osmolality ratios during furosemidediuresis.

Micropuncture

0.50 0.25

0.10

(P/TFOIn 0.05

0.025

% PROXIMAL 25 50 75

* .8

0.0101_

NormalHydropenio (11

Inulin

0.0oo L

FIGURE 2A Plasmato tubule to urine (P/U) inulin concer

dropenia. The lines represent ti tion of filtered water remaini

the puncture site.

% PROXIMAL

25 50 75

(PITF)1 0.5 *

0.25-Furosemide(5) 0.10L Inulin

FIGURE 2B P/TF and P/U during furosemide diuresis. IN

distal tubule because data froi

tions of this segment were

animals.

sorption of

approximately

filtrate, orabout one-third

to the earliest accessible p The

95%o

confidence limit line indicate that between

trate was reabsorbed. It c

thatnearly all of the remaii volume that enters the c

sorbed.

Sodium

reabsorption.

ment of

(TF/P)

sodium

20 proximal samples and

eight animals are shown

and in the upper

portion

seen that the

TF/P

ratios

approximated

unity.

In cc

bule, fluid to

plasma

conco

well below

unity.

The

_slop

for the

relationship

betwe

the puncture site isnot

_sig

% DISTAL URINE zero, indicating no change in this variable along 100 0 25 50 75 100 the distal tubule (Table V). Assuming that

chlo-ride is the predominant accompanying anion, so-dium chloride accounted for about 75 o of the

osmolality.

The lower portion of Fig. 3A, which illustrates fractional5 _{sodium reabsorption, shows that}

ap-proximately

35%o

of the filtered sodium is present at the endof the proximal tubule. Approximately I)

10io

of filtered sodium reached the earliest

acces-| sible portionof the distaltubule. This value is less than halfof the fraction offilteredwaterremaining

fluid (P/TF) and plasma at the same

site,

which indicates that sodium was

ntration ratios during hy- reabsorbed in hypertonic proportions by the

rela-he slopes relating the frac- tively water-impermeable ascending limb.

Signifi-ng [(P/TF) Inulin], and cant net reabsorption of sodium along the re-mainder of the distal tubule could not be detected (0.1 < P < 0.2), as shown in Table V. Nearly

%DISTAL URINE _{all of the remaining sodium was reabsorbed in the}

100 0 25 50 75 100

collecting

duct system since fractional sodium ex-cretion was extremely low in these studies

(less

... than 0.1

%).

Potassium reabsorption. Data obtained from 21 proximal and 30 distal tubules during

anti-inulin concentration ratios diuresis in

eight

animalsare shown in

_Fig.

4Aand

lo slope is given for the Table III. Inspection of the (TF/P)K ratios in

m the early and late por- the upper portion of Fig. 4A reveals values

ap-obtained from different proximating unity in the proximal

_tubule.

Con-siderable scatter is evident in the

potassium

con-centration

ratiosin thedistaltubule

(range

=

0.26-of the_{volumedelivered}

3.43),

but the

_slope

of the line indicates a

sig-of the volume _gme nificant rise along the tubule (Table V). When

fortion

slope

of this

segm

these ratios are factored

by

simultaneously

deter-s for

the slope

of

this

minedTF/P

inulin

concentration ratios,"net

addi-1 and

_13%o.

of

_t~he

fil-

_{tion of potassium (0.13 rising to}

_0.26)

_is

demon-:an

be seen i

_Fig.

_2A

strated (Fig.

4A,

lower portion, and Table

V).

ning20%o of the filtered Although the (U/P)K ratio

_(Fig.

4A) rises to

)llecting

ducts is reab- quite high levels, the ratio

_{[(U/P)K/(U/P)nl]}

The results of measure- 5TheTF/P or U/P ratio for sodium, factored _by the

concentration ratios in corresponding TF/P inulin ratio, gives the fraction of 31 distal samples from filtered sodium remaining at the puncture site (or final in Tables III and IV

_urinS6

_Since

potassiumis not onlyreabsorbed, but also added of Fig. 3A. It can be to thetubule fluid, the TF/P (or U/P) ratio for this ion, Iin the proximal tubule factored by the simultaneously determined TF/P inulin cntrast, in the distal tu- ratio, is definedasthe amount of potassium relative to the

mntration ratios were all amount filtered remaining at the puncture site. This

re--. . lationship is intended merely to provide a convenient

Me of the regression line means of evaluating the fate of potassium in the tubule !en the

(TF/P)NNa

and by taking into account the simultaneous reabsorption of

TABLE V

Slopes oftheRegressionLinesCalculatedfrom Data Obtained from the Distal Tubule of Hydropenic Monkeys*

Fraction of Fraction of

Iranstubular Fractionof rranstubtflar filtered sodium Transtubular filtered

potas-osmolality filteredwatcr Na conce. remaining K concen. sium remaining ratio remaining ratio (TF/P)N. ratio (TF/P)K

y=(TF/P).om y =(P/TF)in y =(TF/P)N. (TF/P)in y=(TF/P)K (TF/P)In

Slope 0.00070 -0.00249 0.00048 -0.00332 0.0066 0.00485

SE ±0.00102 40.00095 ±0.0017 ±0.00225 ±0.0025 ±0.00219

No. observations 28 33 30 28 30 28

Pvalue 0.5 <0.025 >0.5 0.1 < P < 0.2 <0.025 <0.05

*_{These slopes arecalculated from the relationship log y} =a + b xwhere y represents the value for each heading defined above, and x represents the puncture site expressed as per cent of the total'length of the distal convoluted tubule. A P value of <0.05, by convention, indicates that therelationshipisasignificantone(i.e., there is greater than a95%chance that the slope is different from zero).

% PROXIMAL 25 50 75 1.00 de% . _. goe ,

0.50_ 0.25

-0.10_

0.05_

1.001 0.50

0.25

(TF/P)14e 0.10

(TF/P)10 0.05

0.01 _

0.0 025_

%DISTAL URINE 100 0 25 50 75 100

3.

**

I.

(.011)

.I

0

* *.*0

Normal Hydropenia (8)

Sodium FIGURE 3A Tubule fluidtoplasma and urine

to plasma sodium concentration ratios (up-per) and sodium clearance ratios (lower)

* .0025 during hydropenia.

(Table I) does not increase above the mean value

ofthis ratio inlate distal tubule. Totheextentthat

these surface convolutions of the distal tubule can

be assumedtoberepresentativeof all distal tubules in the kidney, these data indicate that net potas-siumaddition in the collecting ducts didnot occur.

Furosemide

After the administration of this diuretic, urine

flow increased promptly and in all instances reached maximal levels within 15 min. Glomerular filtration rates (Table I) were similar to those obtained during control studies in hydropenic animals. Meanvalues forfractional water, sodium,

(TF/P),b

% PROXIMAL %DISTAL URINE

25 50 75 100 0 25 50 75 100

1.00 c- . .r

0.50 0.25

0.10

1.001l (TF/P) 0.50

(TF/P)I. 0.25

0.10

I.

Furosemide (5)

Sodium A.

FIGURE 3B TF/P and U/P sodium concentration

ra-tios (upper) and sodium clearance ratios (lower) during furosemide diuresis.

twc t-: 0

0 U) %) O 0 lf),

oo m 4 t 4 m 1

.t 8 H~~

U) 8: o. X . o..W

0~~~~~~~~~~~~~~~~~~~~~~~~~~0

00 Itt~~~~~~~~~~~~~~

z~~~~~U

v

u e°°°

00U)o

U)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~j

) *

-4 0 .-' 0 t-- d4C1400

C-4-N~~~

'0 U) uo t . u) _ _

N t .Y A CsU) n N + eCh Ut

XoIC'~ '-4 If) U n 0

0-4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-0

-0 r. .

'0

¢~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

04=C14

U)-~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~08 C)u00 '0~

H~~~~~~~~~~ro0 C1o % - CK o1w m C14 I-e oc

a~e 6B~6 6 66 0 0 oooXoo b o 0

co

IdI W 0% ) 0 0 '0

U) @ d OoOOOOOoOcOoOooO-s 00 0 0

0

8

0o000 '00%0%00Z- 04'0

06~~~~~~~~~~~~~~~~~~~~

Ul)V)C1-4 000U)00'1 d4 00t-%0Olt '-4-00 00 ..40

(z4 oo ~0%' C4 'UC 0\0 v) \00'h -4v-'- -. _0'C1

C14

w~~~~~

CtoxC14 0 to m C\ in m C,4 Cq

I-S~~~~~~~~~~~~~~~~~~~~~~~~~~~l _{Id Id} 0 to tn to

4-0 4U

Aauavo) oo'0o sou)c oo

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C6 0 o o 4 C14 C14 _14 c14 C14 oq m )eo en m _ 444 o.04 cs44 -.14 Z

and potassium excretion in the urine after

fu-rosemide administration are compared to mean

values inhydropenic animals in Table I. The

frac-tion of filtered sodium and water excreted ranged from 18 to 36%o, and 24 to 42%o respectively, whereas fractional potassium excretion ranged from 61 to 108%o. As shown in Fig. 1B, 3B, 4B,

and Table III, proximal TF/P osmolality and sodium and potassium concentration ratios were

unchanged whencompared to studies during anti-diuresis. The slope of the line representing frac-tional water reabsorption in the proximal tubule (- 0.00667±0.00156 SE) was not significantly different from the value found during antidiuresis

(Fig. 2B).

In contrast, events in the distal tubule were

found to have changed markedly. Furosemide nearlyabolished the distal transtubularosmotic and sodium concentration gradients (Fig. 1B, 3B, and Table IV). This increase in solute concentration was present in samples obtained from the early distal tubule, and further changes occurred neither along this segment nor in the collecting ducts [mean (U/P) osm=0.94 + _{0.03 SD]. The} frac-tion of filtered water

(P/TF),1,

remaining in the distal tubule was not significantly different from that found during antidiuresis (Table IV and Fig. 2B), indicating that fractional water reabsorption

to this point was unaffected by furosemide. How-ever,thefraction of filtered sodium in this segment was twice the value found in the control group (Fig. 3A and 3B). Further net sodium reabsorp-tion inthe distal tubule orcollecting ducts was not detected. Although it appears from Fig. 2B that

water reabsorption occurred in the distal tubule, itis not possible to state this occurrence with

cer-tainty, since most of the samples obtained from early siteswerefrom one animal. After furosemide

administration, little additional water reabsorption occurred in collecting ducts (Fig. 2A, 2B, and Table I).

After furosemide administration, all (TF/P)K

concentration ratios inthe distal tubule were equal

to or greater than unity (range=0.98-3.43), as

shown in Fig. 4B upper portion. The amount

of potassium present in the distal tubule relative

to the amount filtered was considerably greater in the furosemide-treated animals (Fig. 4B, lower portion). Although it appears from

'Fig.

4B that there was no net addition of potassium in the

(TF/P) re

% PROXIMAL

25 50 75

100_ 50 _

10_ 4.0

2.0

1.0 , ,^r : @.

05

0.25

I.OCl

(TF/P)5*0.50

(TF/P)1, 0.25

0.10

K

Is

%DISTAL URINE 100 0 25 50 75 100

*

.e,

Normal Hydropenio(8)

Potassium

FIGURE4A Tubule fluid to plasma and urine to plasma potassium concentration ratios (upper) and potassium

clearance ratios (lower) during hydropenia.

4.0

% PROXIMAL

25 50 75 100 0 25%DOSTAL50 75 100 URINE

I r

*

2.0 F ( TF/P)K

TF/P)K,

(TF/P)1i

1.0 , ", * i'% .

0.5-

0.25-1.°1

025

0.25

1-Furosemide (5) POtassium

FIGURE 4B TF/P and U/P potassium concentration

ra-tios (upper) andpotassium clearance ratios (lower) dur-ing furosemide diuresis.

distal tubule, it should be remembered that the samples from the early and late portions of the

distal tubule were not from the same animals.

Similarly it is not

possible

to be certain of the fate of potassium in the collecting ducts, because of thescatterin thedata.

Bicarbonate reabsorption. In Table VI and Fig. 5 are summarized the results obtained in four experiments, in which the TF/P bicarbonate

concentration ratios were calculated from the pH

oftubule fluid determined with quinhydrone elec-trodes before and after administration of acetazol-amide.

Study Nephron

I.. .

.-0

(TF/P)HCo-% PROXIMAL TUBULE 0 25 50 75 100 4.0

-20-0

i-1,_1.0

I~~0

2

0.51-0.25

F

0 o

*BeforeDiomox

oAfterDiomox Bicorbonote (4)

0125L

FIGURE 5 Tubule fluid to plasma bicarbonate concen-tration ratios. After diamox, samples of tubule fluid were

obtained by the recollection technique.

Plasma HCO3 concentrations and pH were rela-tively stable throughout each experiment and changed only slightly after acetazolamide adminis-tration. Note that in Experiment 3 the plasma

HCO,

concentrations were low, but the (TF/ P)]HCO3 ratios were not obviously different from

values obtained in-the other experiments. The bi-carbonate concentrations and pH of all samplesof fluid obtained from the middle third of the prox-imal tubule prior to acetazolamide administration

were significantly lower than those in plasma.

From these data we were unable to detect a pro-gressive decline in (TF/P)Hco3 along the course

of the proximal tubule. However, it can be seen

that the only early proximal sample (P 12%o) had

a bicarbonate concentration almost identical with the concentration in plasma. After an intravenous injection of acetazolamide, nine recollection

sam-ples from three animals revealed a uniform

in-crease in

(TF/P)ico3.

In all but two

instances,

the bicarbonate concentration in tubule fluid

ex-ceeded the concentration in plasma.

DISCUSSION

The study of nephron function in the rhesus

mon-key by use of the micropuncture technique offers several advantages over similar studies in rodents and dogs. One important advantage is that in ex-tending these data to an understanding of renal function in man, phylogenetic differences are less formidable. Moreover, theshort loop of Henleand

the arrangement of the

proximal

and distal

con-volutionson the surface of the

kidney

makealmost

the entire nephron, up to the collecting duct,

ac-cessible to micropuncture. Finally, the monkey is more suitable than other animals for study of the mechanism anid site of action of diuretic drugs,

particularly

with respect to

_changes

iln function of

the distal tubule. The rat is much less responsive

to diuretics than man and has been unsatisfactory

to study for this reason.

Fractional water reabsorption in the

proximal

tubule of the antidiuretic monkey is similar to values reported for the dog and rat from this and other laboratories (1, 16, 17). In earlier studies in rat (18-20) and dog (21, 22), variable but greaterfractional water reabsorption-in the proxi-mal tubule was found than in the monkey. This fact may be attributable to differences in the physiological state of the animals, particularly in the extracellular fluid volume. The fraction of filtered water arriving in the early distal tubule

of the monkey is similar to the fraction found in the dog (1). The values reported for the rat (20. 23) are somewhat lower than in the dog and monkey, but again may reflect the physiological

state of the animals studied. In the rat, fluid fromi the early distal tubule is hypotonic and during antidiuresis becomes isotonic to plasma in the midportion (24). During this osmotic equilibra-tion, nearly three-fourths of the volume is reab-sorbed (19, 20). The remaining fraction of fil-tered water (8-10%o) enters the collecting ducts where it is almost completely reabsorbed (2). In the antidiuretic monkey, as in the dog (25), fluid remains hypotonic to plasma throughout the distal tubule. Despite this considerable driving force for passive water reabsorption, only about one-third ofthe volume is reabsorbed. Thus, as in the dog, a larger fraction of the glomerular filtrate enters the collecting ducts of the monkey than enters the

collecting ducts of the rat (1).

The reabsorption of sodium in hypertonic

pro-portionis in the

loop

of Henle is evidence that the primate nephron has the potential to generate the "single effect," the process fundamental to the

countercurrent mechanism for urine concentration (26). However, it should be remembered that cortical nephrons, such as those studied here, do

net sodium reabsorption in the distal tubule be detected,presumablybecause of the low permeabil-ity to water and the presence ofa limiting concen-tration gradient for sodium (1). As in the rat, the site of the steepest transtubular concentration gradient for sodium is in the collecting duct, not

the distal tubule (2).

On the basis of clearance data (27) it has been

shown that in the dog, furosemide administration

in doses of 0.5-5.0 mg/kg impairs free water

ex-cretion and reduces free water reabsorption under

appropriate experimental conditions. Since the

ascending limb of Henle's loop is the

only,

site in the nephron where sodium

reabsorption

par-ticipates in the formation of both dilute and

con-centrated urine, it has been concluded that the principal site of action of this drug is in this seg-ment. Inthe samestudythe authors infer from the magnitude of the diuresis (up to 38%o of filtered

sodium) that reabsorption in the proximal tubule was also inhibited. In a micropuncture

study

in rats, Deetjen (28) found an inhibitory effect of furosemide on reabsorption of fluid in the prox-imal tubulewhen GFRwas reducedby about50%,

but he could not detect an

inhibitory

effect when GFR remained in the normal range. In another micropuncture study,Dirks, Cirksena and Berliner (29), using the recollection micropuncture

tech-nique, were unable to detectan action ofthis drug

in the proximal tubules in dogs. Rector and his

associates (16), usingboth stopped-flow and free-flow micropuncture techniques in the rat, found that although furosemide inhibited intrinsic

reab-sorptive capacity by about 40%o, the drug had no

effect on fractional reabsorption in the proximal

tubule. The failure to detect a decrease in frac-tionalreabsorption wasthought tobedueto a dis-proportionate rise inthe volume of the tubule rela-tivetothe filtration rate, as evidenced bythe pro-longed transit time observed after administration

of the drug. In the present study, furosemide ad-ministration in doses comparable to those used in previous experiments resulted in the prompt excre-tion of upto36%o ofthe filtered sodium and upto

42%o

of the filtered water. Despite this massive

diuresis, fractional reabsorption in the proximal tubule was not depressed. Since the filtration rate was also unchanged, the absolute rate of sodium reabsorption in the proximal tubule was similar to

controlvalues. The fraction of filteredwater

reach-ing the accessible portion of the distal tubule did

not differ from the control value. However, frac-tional delivery of sodium to the distal tubule after furosemide administration was twice that found in controls, indicating that the inhibition of sodium reabsorption produced by this drug occurred at a

site or sites beyond the water permeable portion

of the nephron, i.e., beyond the descending limb of Henle'sloop.

The interpretation of the TF/P ratio for potas-sium depends on a consideration of the electrical

potential. A recent study of the electrical potential

in the rat nephron by Frdmter and Hegel led them to the conclusion that there is no electrical poten-tial difference between the proximal tubule lumen and the surrounding interstitial fluid (30). Their primary observations were essentially identical with those obtained by other workers who have measured the electrical potentials in the proximal

convoluted tubules of rats (2, 31, 32) and dogs (33). When the tubule is first punctured a nega-tive deflection is encountered; in a large number of studies, these negative deflections have always varied rather widely but have generally averaged close to 20 mv, tip of the exploring electrode nega-tive. This potential may be maintained for a pe-riodvarying from a fewsecondstoseveral minutes, generally shorter in the dog than in the rat, and then drops to zero. Most investigators have as-sumed that the 20 mv value represents the true transtubular potential, and that the drop to zero is the result of short-circuiting through the punc-turehole. However, Fr6mter and Hegel found that the potential difference remained at zero even when the tip of the exploring electrode was thrust down the lumen far enough so that any electrical leak at the puncture site should have had little effect on the observed transtubular potential. They concluded that the zero value was the true value, andthat the substantial negative value initially ob-served is derived from some intracellular location, presumably the brush border.

Since allpreviousobservers have found both the

20mv and the zero potentials, it has seemed to us

unprofitabletoexplore the problem with additional similar measurements. The problem is one of

choosing which of the two populations represents

the true transtubular electrical potential. In a recent extensive series of measurements in the dog (1) and in the present study in the monkey, the

potassium concentration in the lumen of the prox-imal tubulehas been found to be very nearly

idein-tical with the concentration in plasma, with only small variations in the mean value. This fact leads us to extend the conclusion of Fromter and Hegel to the dog and monkey, to infer that there is prob-ably no significant transtubular electrical potential difference, and further to infer that the reabsorp-tionofpotassiuminthe proximal convoluted tubule is probably passive, with the potassium ion close

to diffusion equilibrium across the tubule wall. Theseconclusions derive from the fact that if there were an electrical potential gradient and if potas-sium were passively distributed, the concentration ratio would not beone. On the other hand, ifthere

were active transport of potassium, with or

with-out an electrical potential

difference,

the

concen-tration ratio of one wouldnot be more

likely

than any other value. We therefore believe that the observations are most easily explained if it is

as-sumed that there is neither an electrical gradient

nor active transport of

potassium

in the proximal convoluted tubule.

The conclusion that

potassium reabsorption

in the proximal convoluted tubule is passive is at

variance with the conclusions of several previous

studies in which TF/P

potassium

concentration ratios significantly above and below one were

found (and in which a

significant

transtubular electrical potentialwasassumed)

(31-35).

Weare

unable to explain the difference between the

con-centration ratios found in this

study

and previous

studies. However,we areconfidentthatthe

analyt-ical procedure used in the present

study

is

con-siderably

more reliable than that used in the earlier study in the dog (34) and

probably

that used inmost ofthe othersas well.

A (TF/P)K less than unity in the

proximal

tubule has been found in the presence of a

high

concentration of a poorly reabsorbable

solute,

such

as during the administration of mannitol

(2, 35),

or during microperfusion of the tubule with

iso-7This argument does not applyto the concentration of

sodium. The concentrationofsodiumin the lumenmust

re-main close to the concentration in plasma so long as

so-dium salts constitute all but a minor part of the solute

in the lumen and the fluid in the lumen has the same

osmolality as the plasma. Potassium contributes so little

to the osmolality of proximal tubule fluid that its

con-centration can vary independently of the osmolality.

tonic raffinose solution (34, 36). However, those data do not add to the evidence for an active process for reabsorption of potassium, since the

same investigators found similarly low

_(TF/P)K

ratios in the proximal tubule in the absence of a poorly reabsorbable solute, (i.e., in free-flow col-lections during hydropenia).

About one-eighth of the amount of potassium filtered appeared at the earliest accessible portion of the distal tubule of the monkey. It cannot be resolved whether this potassium represents a por-tion of the original amount filtered or potassium

newly added by the inaccessible loop and early distal tubule. Net addition of an amount of potas-sium equal to that initially present in the distal tubule was found in this study; no further addition was found to occur in the collecting ducts. Similar observations have been reported in the rat (2). If the electrical potential in the distal tubule of the monkey is negative with respect to peritubular fluids, as it is in the rat, then the observation that the (TF/P)K in the early distal tubule is less than one indicates, by the usual criteria, active reabsorp-tion of this ion at or just before this site. Furose-mide appeared to inhibit this reabsorption. Similar results have recently been reported for the dog (1).

The reabsorption of bicarbonate in the proximal tubule of the rhesus monkey is nearly complete, a

findingsimilar to that in the dog and rat (21, 37). The pH of tubule fluid in quinhydrone microelec-trodes was measured at a time when the bicar-bonate was in equilibrium with carbon dioxide at a tension approximately equal to that found in extracellular fluids. If a similar equilibrium exists in the lumen of the proximal tubule, as has been suggested by Rector and his associates (38), then the pH recorded in these experiments in vitro (7.04) will be approximately equal to the true pH in vivo. This degree of acidification of proximal fluid is similar in the rat (37), and considerably more than has been found recently in the dog under similar experimental conditions (39). After

administration of acetazolamide, the bicarbonate

concentration in the tubule fluid rose, often to a value exceeding the concentration in plasma. This

indicates thatreabsorption ofthis ion was inhibited

to agreaterdegreethan the reabsorptionofsodium

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